Project Summary/abstract Background: Our work has established that the Ron receptor tyrosine kinase is a key mediator of breast cancer metastasis in humans and mice, but the mechanisms of Ron function in metastasis are still unclear. We recently discovered that the key function of Ron in metastasis is not in the tumor itself, but is in the host immune system. Activation of Ron in the host allows metastatic tumor cells to escape immune surveillance ? facilitating development of metastases. Genetic deletion of the Ron kinase domain specifically in the host, or pharmacologic Ron inhibition, prevented outgrowth of metastases by promoting a CD8+ T cell response that killed metastatic cells. Interestingly, Ron expression in the immune system is restricted to resident (not bone marrow-derived) tissue macrophages, indicating that these specialized cells have an under-appreciated role in regulating metastasis of tumors through immune regulation. We envision that resident macrophages are ?first responders? to the disruptive insult of early metastatic colonization and/or growth, and that they may play a fundamental role in shaping subsequent immune responses. We propose that Ron regulates an immune- suppressive switch in resident macrophages during metastasis, and that inhibiting Ron reinstates immune- mediated killing. Our preliminary data indicate that Ron upregulates expression of the PD-L1 T cell checkpoint ligand and tumor-promoting cytokines in resident macrophages. Indeed, preliminary results show that combined inhibition of Ron with a complementary T cell checkpoint inhibitor, anti-CTLA4, is extremely effective in stimulating immune responses and eradicating tumors. We hypothesize that that Ron-expressing macrophages suppress CD8+ T cell function through a MAPK-dependent immunosuppressive program regulating PD-L1 and tumor-promoting cytokines. We will test this hypothesis through execution of three specific aims: First, we will determine if the Ron inhibitor ASLAN002 improves response to anti-CTLA-4 immunotherapy in metastatic breast cancer models. Second, we will determine if myeloid-specific deletion of Ron kinase blocks metastasis of mammary tumors. Third, we will determine if Ron regulates CD8+ T cell suppression through MAPK signaling and upregulation of PD-L1. Understanding the biology of how Ron functions in the immune response to tumors, and how Ron inhibitors can be useful in the treatment setting, is critical to optimize clinical trial design and maximize benefit to patients. The Ron inhibitor ASLAN002 is in trials and well tolerated, so our work can have near-term impact.